Efficient multiple stage screening method to eliminate pathogenic infection in farmed animal populations

ABSTRACT

Animal husbandry has always been susceptible to the ravages of pathogenic infections. Poultry flus and cattle diseases are but two examples that have dire consequences for animals and adversely affect the economic fortunes of farmers. A testing and culling methodology is presented that can eliminate pathogens from an infected herd. The sensitivity of PCR to detect very low levels of nucleic acid of an infecting pathogen is utilized to identify infected animals. In addition, it has been discovered that PCR analysis of manure samples can accurately identify infected animals and offspring for those species that consume placental remains after birth. Mink Aleutian Disease Virus (mADV) is one of several deadly DNA parvoviruses that can quickly reach epidemic proportions in a mink herd. PCR primers have been developed that generate amplicons to detect and identify the mADV. In addition, a previously unidentified strain of mADV has been discovered, genomically sequenced, and substantially detailed.

This application hereby claims the benefit of U.S. ProvisionalApplications No. 61/274,828 and 61/274,829 filed on Aug. 21, 2009 andU.S. Provisional Application No. 61/286,885 filed on Dec. 16, 2009.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted viaEFS-Web and is hereby incorporated by reference in its entirety. TheASCII copy, created on Dec. 15, 2009, is named 3201-200.txt, and is53,851 bytes in size.

I. BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention provides a method to identify and remove pathogeninfected animals from a group/herd to prevent the spread of infectionand preserve the health of the animals. In particular, PCR screening,utilizing primers appropriate to the pathogen, of both the animals andtheir environs unambiguously identifies active infections.

B. Description of Problem and Prior Art

Pathogens that infect farmed animals affect both the health and survivalof the animals as well as the income of the farmers who raise theanimals. For many pathogens, antibiotics are administered to the animalson an intermittent or continuing basis. However, the presence of theantibiotics or their by-products in consumable food products has raisedconcern about their long-term effect on human and animal health.Immunization against some pathogens is another possible approach, butvaccines for many animal diseases are either not available or are notcost effective. Yet, for other pathogenic organisms no antibiotic orvaccine treatment is available. Early detection of the infection andelimination/removal of the infected animals is the only method that canbe used. However, serologic detection methods vary in their sensitivityespecially during the early days of infection and may only detect aninfection after the animal has started to make antibodies to thepathogen and may, itself, already be infectious.

One pathogen for which there is no effective treatment and no availablevaccine is the pathogenic mink Aleutian Disease Virus (mADV). This viruswas first described in 1956. All mink Aleutian Disease Viruses aresingle stranded DNA viruses of the parvovirus family. There are manystrains of the virus, but only one known non-pathogenic strain (strainG) while the others are typically fatal. The pathogenic viral strainsare absolutely devastating to mink farmers spreading quickly throughmink colonies and contaminating the farm site through contact with themink and their urine and feces. These viruses typically elicit ahyperimmune response in the mink with lethality arising from macroimmuno-antigen complexes. The hypergammaglobulinemia condition inflamescirculatory filtering organs such as the kidneys (glomerulonephropathy),spleen, and liver causing failure of these organs and death from thecomplications.

Attempts to find treatments for parvovirus infections have beenreported. Alvarez et al. in U.S. Pat. No. 5,785,974 suggests that animmunogenic peptide in conjunction with other immunogenic complexes canbe used to make a vaccine that can protect dogs, cats, pigs, and minks.However, the vaccines are proposed to be useful only against anotherparvovirus infection in mink, Mink Virus Enteritis (MVE) not the MinkAleutian Disease Virus (mADV). Barney et al. In U.S. Pat. No. 6,054,265describe peptides that can be used both for screening for certainviruses and for possible treatment. Among other viruses are listed theMink Virus Enteritis (MVE) and the Aleutian Mink Virus (strain G). Thepatent basically deals with HIV identification and possible treatmentmethods are suggested for clinical treatment of infected patients. Nodirect application to infection with the deadly form of the Aleutianmink virus is discussed. Elford et al. In U.S. Pat. No. 6,248,782 teachthat polyhydroxy benzoic acid derivatives are useful in the treatment ofdiseases caused by retroviruses as well as in the treatment of diseasescaused by DNA parvoviruses. No specific example of treatment for minkAleutian disease is given. As far as is known, none of the abovesuggested approaches to containing a fatal mink Aleutian diseaseoutbreak has been successfully employed.

The inventive methods disclosed in this patent document are exemplifiedby the detection and eradication of pathogenic mink Aleutian diseasevirus from a farmed mammalian herd. However, the methodological approachtaught here is applicable to detecting and eradicating pathogens fromany farmed mammalian herd.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the number of mink deaths per week on a Pennsylvania farminfected with Aleutian mink disease for the years 2006 through December2008.

FIG. 2 is a photograph of a typical electrophoresis gel showing thelocations of the GAPDH and mADV marker amplicons.

FIG. 3 is the contiguous partial sequence corresponding to the StahlmADV strain starting at approximately 272 bp and ending at approximately4440 bp of the G strain (SEQ ID NO: 17).

FIG. 4 shows the DNA sequence of the ADV G-strain (SEQ ID NO: 18)alongside the contiguous partial DNA sequence of the Stahl mADV strain(SEQ ID NO: 19) so far determined. The alignment was obtained usingClustal W alignment utility located athttp:///www.ch.embnet.org/software/ClustalW.html. Primers that workedare shaded while primers that did not work are underlined. Thehypervariable region is underlined and identified.

FIG. 5A is the amino acid sequence (SEQ ID NO: 20) of one proteinspecified by the Stahl mADV that does not include the hypervariableregion. This protein is found at the same region of the genome as aprotein found in the G strain.

FIG. 5B is the amino acid sequence (SEQ ID NO: 21) of a second proteinspecified by the Stahl mADV that does include the hypervariable region.This protein is found at the same region of the genome as a proteinfound in the G strain.

FIG. 6 is a comparison of the partial amino acid sequences of severalknown mink Aleutian disease viruses aligned (SEQ ID NOS 22-31,respectively, in order of appearance). The hypervariable region is boxed(boxed sequence in StahlX1 disclosed as SEQ ID NO: 32).

FIG. 7A is an outline of the screening method of the inventionindicating the type of test applied at each stage and the disposition ofanimals that tested positive and negative.

FIG. 7B is an outline of an embodiment of the screening method of theinvention in which PCR mADV screening, but not antibody detection, inblood is used.

FIG. 7C is an outline of a preferred embodiment of the screening methodof the invention in which the herd is retested by PCR screening duringthe period roughly from December to February.

FIG. 7D is an outline of a preferred embodiment of the screening methodof the invention in which additional PCR testing of fecal material isperformed at the time of whelping.

FIG. 7E is an outline of a possible method to identify and placenon-permissive animals into a breeding herd.

FIG. 8A is a photograph of an electrophoresis gel showing the result ofa composite placental manure PCR identification of mADV infection.

FIG. 8B is a photograph of an electrophoresis gel showing on the leftthe result of PCR screening of the four females from the compositeplacental manure sample of FIG. 8A. None are mADV positive. Theoffspring of three of the four females were all mADV negative. On theright of FIG. 8B is the result of PCR screening of the 7 offspring ofthe fourth female from the composite placental manure sample of FIG. 8A.PCR identified three of the seven offspring as mADV negative while fourof the seven offspring were PCR positive for mADV.

FIG. 9 shows the number of mink deaths per week on a Pennsylvania farminfected with Aleutian mink disease for the years 2006 through September2009.

DETAILED DESCRIPTION OF THE INVENTION

A. Characterization of a Rampant Epidemic Infection and Need for aSolution:

The consequences of mADV infection, both in terms of animal survival andof economic survival of the farmer, are extreme and a solution to theproblem is urgently needed. Just how extreme the consequences are ishighlighted by the experience of the inventors. As noted above, deadlymink Aleutian Disease Virus (mADV) infection can quickly spread througha herd with devastating consequences. FIG. 1 shows the number of minkdeaths per week on a Pennsylvania mink farm run by the inventors thathad previously been virus free. Prior to June 2006 relatively few deathsoccurred generally arising from environmental stress on the herd. Eachlineage of minks had been raised on the farm for at least 35 years. InMay 2007 health problems in the herd were first noted with some animalshaving bleeding gums and blood infused water cups. E. coli was ruled outand mink ADV was considered a remote possibility since the farm had beenmADV free since a mild strain was eliminated by standard husbandrytechniques alone in the late 1960's. However, CIEP(counterimmunoelectrophoresis) testing on Jun. 12, 2007 indicted thatapproximately 30% of barren females were mADV positive.

Despite an extensive testing and animal segregation program using ablood antibody detection procedure (LFIA dipstick—Scintilla Development,Bath, Pa.) the infection continued to spread. Emptied pens that hadcontained positive animals were disinfected with Kennel Care, reportedlya broad spectrum parvocide. However, animals later transferred to thesepens had a 90% reinfection rate, and it was concluded that thisparvocide was not effective against mADV. By the end of September andthe beginning of November, 2007 approximately 130-150 animals were dyingper day as illustrated in FIG. 1. By the end of 2007, the herd had beenreduced from roughly 14,000 members and 3,000 breeders to 7,000. At lest50% of the mink died, another 30% were symptomatic, while 15-20%appeared asymptomatic. The disease spread was unstoppable.

One choice for the 2008 raising season was to dispose of all the animalsand start the herd with imported healthy animals. However, this wouldhave meant losing decades of selective breeding and a unique gene pool.In addition, important value would be gained by keeping the naturallyresistant mink that survived the epidemic. Realizing the inadequacy ofthe testing methods, for the 2008 season only 3,000 asymptomatic femalebreeders were kept with no further testing. However, 1,000 of the 3,000animals were lost by March, 2008 and about half of the remaining 2,000females never produced surviving offspring. Of the other 1,000 females,600 produced diminished litters of 3 or less and 400 produced litters of4 or more. These 400 animals and their litters were kept but those thatsubsequently became symptomatic were removed. By late July 2008 it wasobvious that an accurate method of virus detection was urgently needed.

B. Development of PCR Based Virus Identification:

The problem for herd management with known antibody testing methods isthat the tests detect antibodies produced only after the animal hasmounted an immune response some significant amount of time afterinfection. In addition, the virus persists outside of the animals. Threetests had been in common use in herd management. IAT (iodineagglutination test) is non-specific for mADV and detects only 16-65% ofpositive CIEP reactors. It is not possible to eliminate mADV from theherd by culling with this test (Gorham, Henson et al., Infection [1976]pp 135-158). CIEP sensitivity is uncertain below antigen titers of 8-16.However, a false negative window exists for at least one weekpost-infection, and CIEP will not determine if the virus was eliminatedfrom the host. The best results for CIEP (0.5-3.2% positive reactors)were determined 1 year post test. (Cho, Greenfield, J ClinicalMicrobiology, January [1978] pp 18-22). If time and resources areavailable, post exposure antibodies can be detected at a fairly earlystage using an ELISA assay (enzyme linked immunosorbent assay). Underfarm conditions where a large number of animals (hundreds to thousands)need to be screened, a LFIA strip (lateral flow immunoassay) may be usedin place of ELISA. Finally, ELISA is consistently more sensitive thanCIEP (95% vs. 65% or less) (el-Ganayni, Pub Med, [1992] pp 134-151) andis a rapid cost-effective method of detecting exposure to mADV. However,there exists a false negative window for three weeks post-infection.Further, the false negative rate experienced with LFIA can range from4-14%. The test will not determine if the virus was eliminated from thehost.

If possible to implement, clearly the best alternative available wouldbe testing the animals for the presence of the nucleic acid of the mADVvirus using Polymerase Chain Reaction (PCR). PCR can detect virus daysafter infection and at a very low level (less than 1 femtogram—about 10genomes of ADV DNA in 2.5 μL of serum (Durrant, Bloom et al., JVirology, February [1996] pp 852-861)). There is the possibility offalse negatives due to sequestration of the virus, and, for this reason,the test will not determine if the virus was eliminated from the host.However, the ability to unambiguously detect the presence of the virusmakes PCR the best choice for monitoring a herd and eliminating theviral infection.

Unfortunately, as of approximately July 2008 no laboratory wasimmediately available to perform a PCR test for mADV particularly on thescale required (several thousand animals) and at a non-prohibitive cost.Further, most importantly, at that time it was unknown whether a PCRtest existed that could detect the strain of mADV infecting theinventors' herd.

(1) Discovery of Appropriate Primers:

In order to develop primers suitable for PCR testing of the infectiousmADV, the nucleotide sequence of the non-pathogenic Aleutian mink virusG strain was examined. This sequence had been published by Bloom. Thenucleotide sequence was obtained from PubMed.com (NCBI ReferenceSequence NC_001662.1). In addition, primers to a universal target, minkglyceraldehyde 3-phosphate dehydrogenase (mGAPDH), were developed. ThemGAPDH nucleotide partial sequence (Gram-Nielsen, et al.) was obtainedfrom PubMed.com (GenBank: AF076283.1). Multiplex PCR utilizes more thanone set of PCR primers in the same reaction to allow simultaneousamplification of more than one target sequence. In such a controlledreaction, one pair of the multiplex PCR primers is used to detect thepresence of the target in question while the other primer pair acts asan internal control to a universal target and assures that the qualityof DNA extracted and PCR condition/technique is successfullyimplemented. Multiplex PCR was used for all testing for the mADV.

The entire G strain sequence was entered into PrimerQuest (IntegratedDNA Technologies, idtDNA.com), and possible primers identified followingsuggestions by the Integrated DNA Technologies' on-line IDT SciToolsapplication. Approximately 50 different primer pairs were suggested. Abest guess was made for the first primer pair to be tried and theprimers were ordered. Astoundingly, the first primer pair attempted,V3-F/V2R worked and yielded an amplicon of ˜378 bp. Because thisamplicon size was too close to the GAPDH amplicon size to clearlyresolve on the electrophoresis gel, another primer pair, V3-F/V3-R, wastried, and it also worked and yielded a large amplicon easilydistinguished from GAPDH. Shortly after this success, primers that spanthe hypervariable region (which was previously known by Bloom) weresought in order to identify the particular mADV strain infecting theherd.

Once primers were identified that covered the hypervariable region, thesequence of the hypervariable region was obtained. It was quicklyrealized that the mADV strain on the inventors' farm did not correspondto any strain in the published literature and was, therefore, a novelstrain. The sequence of the Stahl mADV genome was determined asindicated below in Section B (2). Subsequent to the initialidentification of the first primer pairs, portions of the G strainsequence were entered into PrimerQuest and possible primers suggested.Selection of several primer pairs were made based on a judgment of whatmight work. Other primer pairs were tried over a course of about tenmonths in order to both identify the best primers to use to detect mADVand to identify primers having a sufficient coverage over the genome inorder to sequence the entire virus genome. As is well known, primerselection is still an art and not an exact science and much trial anderror was involved in determining useful primers. Some of the primerpairs worked while others did not, possibly due to mutual inhibition orto the inability of a particular region to anneal well. Primer pairswere suggested by PrimerQuest based on “relative abilities” to work as aprimer based on the input sequence (or partial sequence). The remainingportions of the G-sequence were entered this way to find primers in theremaining untried regions. The oligonucleotide primers themselves wereobtained from Integrated DNA Technologies. The DNA extraction conditionsfor PCR utilized by the inventors are set forth in Appendix “A”. The PCRreaction conditions utilized by the inventors are set forth in Appendix“B”.

Table 1 lists several of the primer pairs generated, tested, and used inmapping and diagnostic screening based on the G-strain sequence. Thosewith amplicon sizes listed indicate that the pair worked well. As shownbelow, five of the primer pairs that were tried and expected to workhave zero size indicating the pair did not work. The start and endpositions numbers referenced correspond to the G strain sequencepositions.

TABLE 1 Start Conserved Conserved Primer Amplicon (bp End Match Match(For/Rev) Size (bp) position) (bp position) (forward) (reverse) V1/V0 018 381  ?/24 23/24 V1/V1 0 18 932  ?/24 23/24 V1a/V1a 954 273 1227  ?/2423/24 V2/V2 934 895 1829 24/24 21/24 V3/V2 378 1451 1829 23/24 21/24V3/V3 883 1451 2334 23/24 24/24 V4/V4 981 2064 3045 23/24 24/24 V4a/V5a0 2356 3325 23/24 21/24 V4b/V5b 999 2525 3524 24/24 24/24 V5/V5 802 30223824 24/24 24/24 V6/V6 0 3742 4766 21/24  ?/24 V6a/V6a 881 3559 444023/24  ?/28 V7/V6 0 4223 4766 25/26  ?/24The oligonucleotide sequences of some of the above primers used include:

V1a: (SEQ ID NO: 1) 5′ - TTA ACG ACG GTG AAG GAG TTG CCT - 3′ (forward)(SEQ ID NO: 2) 5′ - TCT TCT GGA GTA AAG CAA CCA ACG - 3′ (reverse) V2:(SEQ ID NO: 3) 5′ - TGG TTA CTT TGC TGC TGG TAA CGG - 3′ (forward)(SEQ ID NO: 4) 5′ - TCC TCT GTT TAA GTG GCT CTG CGT - 3′ (reverse) V3:(SEQ ID NO: 5) 5′ - ACC ATC CTA ACC AAG CAA GGT GGA - 3′ (forward)(SEQ ID NO: 6) 5′ - ACA CGT GTC TTG GAG CAC TTC TCT - 3′ (reverse) V4:(SEQ ID NO: 7) 5′ - TGC CAC AAC TGC CAC GAA GAA TAC - 3′ (forward)(SEQ ID NO: 8) 5′ - ATT GGG TTG GTT TGG TTG CTC TCC - 3′ (reverse)V4b/V5b: (SEQ ID NO: 9) 5′ - CAG CAC TGG CGG CTT TAA TAA CAC - 3′(forward) (SEQ ID NO: 10) 5′ - ACT ACC CTG TAA CCC TGC TGG TAT - 3′(reverse) V5: (SEQ ID NO: 11) 5′ - GGA GAG CAA CCA AAC CAA CCC AAT - 3′(forward) (SEQ ID NO: 12) 5′ - TTC AAA GTG TGT GCC TGA AGC AGC - 3′(reverse) V6a: (SEQ ID NO: 13) 5′ - CAA CCA AAG GTG CAG GTA CAC ACA - 3′(forward) (SEQ ID NO: 14) 5′- GGA AGT ACA CAG TAT TTA GGT TGT TCA C - 3′ (reverse)The primer pair used for mGAPDH is:

(SEQ ID NO: 15) 5′- AAC ATC ATC CCT GCT TCC ACT GGT - 3′ (forward)(SEQ ID NO: 16) 5′ - TGT TGA AGT CGC AGG AGA CAA CCT - 3′ (reverse)

As noted above, an initial attempt at diagnosing the presence of mADVvia PCR utilizing primer V3 forward paired with V2 reverse yielded anamplicon of 378 bp. The size of this amplicon was too similar to themGAPDH amplicon of 250 bp to be reliably separated on theelectrophoresis gel. Therefore we ultimately chose an alternative mADVprimer pair (V5) which would yield a larger amplicon (802 bp). Thisresolution was sufficient to clearly distinguish the mGAPDH and mADVamplicons. FIG. 2 is a photograph of a typical electrophoresis get andshows that the GAPDH and mADV amplicons are well resolved and separated.In addition, the V5 primers spanned the hypervariable region of the mADV(Bloom, et al.). This not only yielded an amplicon distinguishable fromthe mGAPDH amplicon, but also enables the strain typing of the virusesby subsequent sequencing of this amplicon from different viruses The V5primer pair represents the preferred enablement and is used routinely asthe diagnostic screening tool of choice for mADV.

As will be readily evident to those skilled in the art, in addition tothe primer pair sequences listed above, the reverse complement sequencesof the above forward primers could also work as reverse primers (i.e.reverse complement of V5 forward equals V4 reverse). Similarly thereverse complement sequences of the above reverse primers could alsowork as forward primers (i.e. reverse compliment of V4 reverse equals V5forward). (This is easily seen illustrated in FIG. 4.) In both theseexamples, a new primer companion would have to be selected because thedirection of amplification would now be different. All primers disclosedshould also function properly if at least approximately 85% of the basesare identical to the primer sequences identified and appropriatelymatched with a primer pair under slightly different annealingtemperatures. As is evident to those skilled in the art, the disclosedprimers should also function properly if 1 or more bases were added tothe 5′-end and 1 or more bases truncated from 3′-end and similarly when1 or more bases were added to the 3′-end and 1 or more bases truncatedfrom 5′-end (when referenced to the G-strain sequence). In addition, aswill also be readily evident to those skilled in the art, any nestedprimers, being a subset of the target region of the described primers,are included in the scope of this disclosure as are other primer pairsthat overlap or are immediately adjacent to the primers described indetail above.

(2) Sequencing of Mink Aleutian Disease Virus:

A novel mADV strain has been identified based on DNA sequences obtainedfrom mADV amplicons produced from the PCR reactions using the aboveselected primers. Amplicons were sent to GeneWiz (GeneWiz, Inc., SouthPlainfield, N.J.) for DNA sequencing. Overlapping DNA segments wereassembled using DNA Baser software (dnabaser.com) to form a contiguoussequence. This sequence was compared to the only published full-lengthsequence G-strain mADV (Bloom, et al.) obtained from PubMed.com (NCBIReference Sequence: NC_001662.1) by the use of Clustal W software(npsa-pbil.ibcp.fr) and determined to be a contiguous partial sequencethat starts relatively around 272 bp and ends around 4440 bp out of the4801 bp total.

Table 2 illustrates the relative alignment positions and sizes of themADV amplicons used to sequence the mADV genome in relation to theG-sequence (vertical bars). Progression over time is indicated from topto bottom starting with V3/V2 and ending with V7/V7. Hatched trellisregions indicate the part of the mADV DNA sequence obtained using thedifferent primer pairs. Region 2.8 kb (horizontal bars along the top ofthe table) indicates the relative hypervariable region (3096-3134 bp).The assembled mADV contiguous region is depicted in the bottom row andwas obtained from overlapping DNA sequences (273-4440 bp). It wasassembled without any gaps by the use of the overlapping ampliconsdesigned by proper primer pair placements. This is considered a partialsequence in relationship to the entire G-sequence since approximatelythe first 272 bp at the 5′ end and 361 bp at the 3′ end have not yetbeen identified.

TABLE 2

The primer pairs that span the hypervariable region are V5-F/V5-R andV4b-F/V5b-R. The contiguous partial sequence of the Stahl mADV strain ispresented in FIG. 3. While the standard procedure of starting thenumbering sequence at “1” has been utilized in FIG. 3, as noted above,the Stahl mADV sequence is a contiguous partial sequence starting about272 bp in from the start of the G strain sequence.

A comparison of the nucleotide sequences of the G-strain and the Stahlstrain is shown in FIG. 4. The alignment information shown in FIG. 4 wasgenerated using the Clustal W alignment utility located athttp://www.ch.embnet.org/software/ClustalW.html. The strainidentifications, numbers, and primer designated sites have been added tothe Clustal W comparison. The primers that worked are shaded, while theprimers that did not work are underlined. The hypervariable regionstarting at 3096 (G-strain reference) is labeled and underlined. ThemADV contiguous sequence was BLAST searched against all other publishedsequences and no other identical match found (PubMed.com). The mADVsequence shown in FIGS. 3 and 4 is the first time identification of thesequence of the highly infectious mADV virus has been determined.

In particular, it will be appreciated by those skilled in the art thatany primer pair that spans the hypervariable region falling within theV5 primer pair including the nucleotide sequence of the hypervariableregion disclosed in this patent document will generate a PCR ampliconspecific to the Stahl mADV strain. Further, since the hypervariableregion specifies the strain type, use of such a primer pair that spansthe hypervariable region with other ADV strains will permit an accuratestrain typing that can be used to not only identify the strain but alsoto trace infections from place to place, herd to herd.

Thus, while other methods of performing PCR have been developed (such asrapid PCR techniques using fluorescence resonance energy transferprobes) that do not rely on electrophoresis gel determinations, any suchtechnique that relies on the amplification or identification of thesequences disclosed in this patent document is considered to beencompassed by the present disclosure.

FIG. 5 shows the amino acid sequence of two proteins predicted from thepartial nucleotide sequence determined for mADV. FIG. 5A shows the aminoacid sequence of one protein specified by the Stahl mADV that does notinclude the hypervariable region. This protein is found at the sameregion of the genome as a protein found in the G-strain. FIG. 5B showsthe amino acid sequence of a second protein specified by the Stahl mADVthat does include the hypervariable region. This protein is found at thesame region of the genome as a protein found in the G-strain. Thesequences were generated using the ExPASy Proteomics Server, SwissInstitute of Bioinformatics (http://www.expasy.ch/tools/dna.html). FIG.6 is a comparison over a limited span of the amino acid sequences ofseveral mink viruses including the G-strain and the Stahl strain. Togenerate FIG. 6, a nucleotide BLAST search was conducted using the Stahlstrain nucleotide sequence as the query on PubMed.com(http://blast.ncbi.nlm.nih.gov). Several similar DNA sequences obtainedwere selected for translation using ExPASy. The resulting amino acidsequences were then aligned using a CLUSTAL W protein alignment utility(http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsaserver.html). The comparative sequences span the hypervariable region(indicated by the box). The amino acid sequence of the Stahl strainclearly differs from the others in several locations. Several differentsingle nucleotide polymorphisms (SNP's) were identified within the Stahlstrain DNA sequence. The differences in the DNA base at these positionseach produce a change in the corresponding coded amino acid. This typeof variant is known as nonsynonymous because a different polypeptide isproduced. Table 3 shows some SNP's identified by the DNA location andresulting change in coded amino acid.

TABLE 3 SNP location (bp) Nucleotide = Amino Acid 301 T > G = H > Q 412A > G = I > M 575 T > C = F > L 908 T > A = C > S 1059 G > C = S > T1068 C > T = T > I 1078 A > C = E > DIt is unknown at the time of drafting this patent document whether theidentified changes are responsible for the virulence of the Stahlstrain. There are indications in the literature that other sites alongthe amino acid chain may also be involved in determining the relativevirulence of the viruses.C. Procedure for Elimination of Pathogens from a Farmed Herd:

In order to eradicate a rampant epidemic infection from a herd, alltesting methods available are used. In the case of a mink farm, both anantibody detection method (ELISA or LFIA) and PCR are used. However,even before animal inspection and testing can begin, a virus free cleanfacility needs to be created so that animals transferred out of theinfected herd are not reinfected. Appendix “C” outlines the sanitationprocedure used on the farm. Importantly, Oxine solution with and withoutadded detergent has been found to be an effective parvovirus viracide.Before cleaning with any product, care should be taken to ascertain thatthat product will inactivate the infecting pathogen. In particular,environmental PCR testing as described in Appendix “E” should beemployed.

Once a clean facility has been obtained, animal selection and testingcan begin. FIG. 7 shows in outline form the methodological sequenceoriginally employed to identify and remove infected animals from theherd. Initially a visual examination of the animals is made to observeany animals showing clinical symptomology. Clinical signs such aslethargy, poor appetite, underweight, ventral staining, discharge fromthe mouth and bleeding gums are all indications but not proof of aninfected animal. Considering the consequences of keeping a potentiallyinfected animal in the herd, no consideration at this time on aninfected farm is given to the actual clinical cause of the observedcondition of the animals. These animals are immediately removed from theherd, and, in the case of mink, are pelted. Only visually asymptomaticanimals are considered for testing. Urine samples are obtained fromthese animals. For mink, urine is collected from a suspended cup placedbelow the animal and above the manure pile. The urine is tested with anantibody detecting method (ELISA may be used but the use of a LFIA stripprovides a quick result and is easily employed in the field). LFIA canonly detect antibodies after the 14-21 days it takes for the animal tomount a sufficient immune response. However, a positive urine antibodytest (using LFIA) on an asymptomatic animal indicates a prolonged andpersistent viral infection and that sufficient renal damage(glomerulonephropathy) has already occurred from antibody/antigencomplexes. Healthy animals will not excrete antibodies in urine unlessthe renal system has deteriorated. The antibody positive animals areremoved from the herd, and, in the case of mink, are pelted.

An antibody negative urine animal is now a candidate for furtherantibody testing of its blood. ELISA or LFIA may be used. Again, asnoted above, LFIA is more conveniently used. LFIA testing of blood is amore sensitive test and does not rely on extensive renal damage havingoccurred. Blood is collected for both antibody testing and PCR testingat the same time according to the method described in Appendix “D”.Whether the blood tests positive or negative for antibodies, the bloodis still subjected to further PCR testing. In the case of an animal withantibody positive blood, it is possible that the animal has acquired anatural immunity to the virus and should be kept in the herd. If theblood PCR test indicates virus present in the antibody blood positiveanimal, the animal is removed from the herd. If the blood PCR testindicates no virus present in the antibody blood positive animal, theanimal is kept in the herd and identified as antibody (+) virus (−). Ifthe blood PCR tests positive for virus present in the antibody bloodnegative animal, the animal is removed from the herd. If the blood PCRtest indicates no virus present in the antibody blood negative animal,the animal is kept in the herd and identified as antibody (−) virus (−).At this point in the selection process, both the antibody (+) virus (−)and the antibody (−) virus (−) animals are kept in the herd.

After some experience utilizing the above outlined protocol, it wasappreciated that nothing was being gained by testing the blood forantibodies. The subsequent PCR test for viral presence is a necessaryand sufficient selection criterion. PCR mADV positive blood testsindicate an infected animal and indicate that the animal should beremoved from the herd. However, as in the earlier protocol whereantibody positive PCR mADV negative animals were not removed from theherd (since the antibody presence probably resulted from the animalnaturally mounting a sufficient immune response to the virus) in therevised protocol PCR mADV negative animals are kept in the herd. Thepreferred protocol embodiment of the invention is outlined in FIG. 7B.

As the animals are characterized and the infected animals removed ordestroyed, the healthy animals are transferred to sanitized pens. Forthis transfer, the animal is caught with Oxine soaked gloves (500 ppm),placed in a small carrier and dowsed repeatedly in a 200 ppm solution ofOxine. Into this solution is also added a small amount of dish washingsoap to aid as a surfactant for the aqueous Oxine solution to penetratethe highly hydrophobic under wool. In this manner, the external surfaceof the animal is treated as completely as possible with Oxine. Oxineaids in the elimination of environmental virus on the mink. It has beendiscovered that it is possible to have a viral blood negative mink in aviral positive pen. Swabbing of the tops and bottoms of pens andanalysis of the swabs by PCR revealed that the top of the pen wasusually more contaminated than the bottom of the pen. In such a pen, avirally negative mink either was not yet infected or the viral load wasnot yet sufficient to cause an infection, but the virus may be carriedon the outside of the body. When a mink from an infected pen is movedinto a clean area, it may unknowingly cause a reinfection at a laterdate. Thus, passing the viral PCR tests is not sufficient to maintain avirus free herd without also sanitizing the exterior of the mink. The200 ppm Oxine solution was not found to have any effect on the eyes ormucus membranes of the mink and is an effective tool for killing thevirus in the mink's coat. Only after undergoing this cleansingmethodology was a mink placed into a freshly sanitized, quarantined,windward area of the ranch.

However, it should be appreciated that it is possible that a recentlyinfected animal may not be detected by PCR testing. Accordingly,retesting of the animals using the preferred PCR protocol may berequired to either confirm the absence of the virus in the herd or toremove any remaining infected animals. Based on the inventors'experience, it is believed that the optimum windows for testing areDecember during pelting, late February prior to breeding, and whelpingseason. PCR retesting according to the protocol set out in FIG. 7C ofthe mink herd on the inventors' farm two months after the abovedescribed testing and selection process discovered that about 1.5% ofthe females and less than 1% of the males were still infected. Inaddition, the pens of these animals were resanitized and left dormant.As can be seen in FIG. 9, the viral elimination protocols outlined abovesubstantially reduced the mink mortality for 2009. It should be notedthat a variety of causes unrelated to mADV infection result in somelevel of mink mortality as is reflected in FIG. 9 for 2009. However, itshould also be appreciated that the viral elimination protocols andhygienic cleaning of the farm result overall in a much healthier herd.

Another method of monitoring the health of the herd has been discoveredusing placental manure screening that will be described below.

D. Procedures for Continued Monitoring of an Animal Herd:

In a large farm consisting of potentially many thousands of animals, thecost in time and expense of utilizing the protocols outlined above foreliminating a contagious infection from the herd is a relatively smallfraction of the loss attributable to the decimation of the herdpopulation. Once a relatively infection free herd is established, otherongoing monitoring protocols can be utilized.

(1) Placental Manure Sampling:

The females of many mammalian animal species, including mink, soon aftergiving birth devour the discharged placenta. Malformed or dead offspringmay also be consumed. The reason for this behavior is not wellunderstood but may be linked to the need for hormones to reduce uterinebleeding (in mammals). In the case of mink, shortly after consumption ofthe placenta, the female mink passes a black, tarry, and shiny stool.Typically the stool is found in a far corner of the pen or even on theground. If deposited relatively soon prior to discovery, the stool iseasily sampled by inserting a small diameter tube a fixed distance intothe medium to collect a sample size of approximately 35 uL. This sampleis placed in a labeled tube for submission for PCR. If the stool hasbeen deposited for some time and the weather conditions are dry, a hardskin begins to form around the medium that must be broken for internalsampling.

Using PCR methods described above to analyze a sample of the stool, ithas been discovered that mADV can be detected in animal manure as shownin FIG. 8A. The placental stool PCR mADV screening enables the removalfrom the herd of the affected dams and litters the day of whelp. Veryimportantly, this method provides a non-invasive and non-tactile methodof screening that does not disrupt the mink during this period withunnecessary handling. In addition, the method minimizes the spread ofthe disease through contact and handling at the beginning of the springand summer (the whelping season), the most contagious times of the year.When a positive manure sample is identified, the animals are removedfrom the herd, and, in the case of mink, euthanized. To reduce thelikelihood of the spread of infection, the litters adjacent to theinfected litter are removed to pens on the leeward side of the ranch forquarantining and observation as an extra precaution. All empty pens arethen cleaned and resanitized as taught previously. Most importantly,since the stool contains material from all the offspring as well as themother, analysis of the stool by PCR discovers infection in theoffspring as well as the mother. Based on the discovery of pathogendetection by PCR in the placental manure of mink, detection of pathogeninfection in the placental manure of other species in which the motherconsumes the placenta may be accomplished by PCR analysis for arepresentative pathogen nucleotide sequence.

On a ranch where the virus has been substantially eliminated accordingto the protocol methods of the present invention, to reduce the numberof manure samples to be analyzed by PCR, sampling of composite birthstools from several animals can be used as an economical and rapidmethod for virus detection. For example, it has been found thatcomposite pooling of samples from four females where one sample ispositive for the virus will reveal the entire composite to be positive.(See FIG. 8A.) FIG. 8A shows the results of PCR mADV analysis of 9different pooled placental manure samples. As shown, the mADV virus wasfound in one pooled sample. Thus the dilution factor of the sample isnot a concern due to the high sensitivity of the PCR method. Higherpooling numbers are possible but the limits have not been explored as ofyet. It is very important to record all members of the pooled sample andthe location of each of the members for future reference should PCR mADVanalysis of individual samples be required. In all cases of a PCR mADVpositive composite sample, the individual samples that made up thecomposite will have to be retested by themselves to find which of thepooled samples had the infection so that the positive animals associatedwith that sample can removed from the herd. Farms that have a history ofthe disease may not be able to afford high pool numbers due to thegreater probability of positive samples.

Screening during the whelping season of the placental manure of allanimals in the Pennsylvania herd after the elimination of infectedanimals according to the protocol methods of the present invention setforth above yielded some interesting results. Two composite placentalmanure samples (four dams in each composite) were PCR positive for mADV.As noted above, FIG. 8A shows the screening result for composite samplesindicating that one composite sample was PCR positive for mADV. PCRanalysis was then applied to samples from each dam and their offspringin the two PCR mADV positive composite pools (not shown). In the firstpositive composite pool, 3 dams and all of their offspring were PCRnegative for mADV. The remaining dam and her offspring were PCR positivefor mADV. Clearly, dilution by composite pooling did not affect theaccurate PCR detection of mADV. The PCR mADV positive animals wereremoved from the herd.

PCR mADV analysis of animals in the second positive pool was surprising.The results of the individual screening for these animals is shown inFIG. 8B. FIG. 8B shows the PCR results for all four females (on the leftof the central ladder column) and the seven offspring of one female (onthe right of the central ladder column). All four females were found tobe PCR mADV negative. Three of the four litters (18 offspring) were alsoPCR negative for mADV (not shown). The fourth PCR mADV negative femalehad a litter of 7 offspring in which 3 of the 7 were PCR mADV negativewhile 4 of the 7 were PCR mADV positive as shown on the right of thecentral ladder column of FIG. 8B. Two very important discoveries comeout of this data. First, PCR testing of the placental manure picked upmADV infection in the offspring. Suprisingly, PCR mADV testing alsorevealed the presence of an otherwise healthy and PCR mADV negativefemale that carried the mADV virus and was capable of passing the viruson to her offspring. Screening by the composite sampling method permitsnot only the identification of infected animals that were kept in theherd having passed the initial screening tests, but, most importantly,also permits the identification of carrier animals that need to beremoved from the herd in order to eliminate all infection from the herd.It is probable that the PCR mADV negative female animal was“non-permissive”; that is, the virus is unable to infect the animal'scells even though virus particles remain sequestered in the animal. Thefemale apparently passed on her “non-permissive” genome to 3 of heroffspring but not the other 4. Prior to this discovery, the relevantliterature has taught that the vertical transmission of disease causedby mink disease virus was 100%. The results shown in FIG. 8B clearlyindicate otherwise.

This is the first indication that there is some genetic variation inmADV susceptibility occurring between generations, and that there is agenetic basis that makes the animals non-permissive. Clearly, all thefetuses develop simultaneously in utero and are simultaneously exposedto the virus, but the virus does not affect some of the fetuses.Interestingly, antibody testing of the non-permissive dam also did notindicate any antibodies. Based on this example, there is a strongsuggestion that a genetic solution to the mADV infection problem may befound. Not only is placental manure screening a cost and time effectiveway to monitor the health of the herd, it is particularly important as away to identify non-permissive animals as early as the whelping day sothat infected animals can promptly be removed from the herd before thereis an opportunity for them to pass on the virus. The full screeningprotocol setting forth the most preferred embodiment is shown FIG. 7D.

In the future, the inventors intend to try to identify the geneticmarkers that are responsible for the “non-permissive” characteristicwith the hope that, with knowledge of the gene sequence identifying thenon-permissive characteristic, a whole herd can be created that isresistant to mADV. Alternatively, the identification of non-permissiveanimals using PCR for mADV on placental manure samples, also raises theinteresting possibility of creating, by breeding, a herd of animals allof which possess a non-permissive genome. At this time it is unknownwhether breeding non-permissive animals with other non-permissiveanimals will produce a stable gene line of non-permissive animals. Apossible alternative scenario for establishing a breeding herd ofnon-permissive animals is set out in FIG. 7E. Instead of pelting thekits that are identified by a PCR mADV positive unpooled manure sample,the kits are individually retested by PCR for mADV. Some of the kitswill test positive since they are the source of the positive manuresample. Any PCR mADV negative kits would be segregated and used toestablish a non-permissive herd. At this point it would be unknownwhether the kits harbor a sequester virus and would transmit the virusto their offspring. Any remaining PCR mADV positive kits as well as thePCR mADV negative dam that is now known to harbor the virus would bepelted. Repetitive identification and segregation of non-permissiveanimals in subsequent generations should establish a gene line thatbreeds true for non-permissive animals.

(2) Saliva Sampling:

Finally, for continued monitoring of the herd, an alternative salivacollection process for PCR can also be employed. Saliva can be collectedfrom mink by allowing them to bite upon a thin plastic tube or string orabsorbent material such that sufficient saliva is collected. No handlingof the animal is required which lessens the transmission of the diseaseand speeds collection. Typically this sampling is best achieved justprior to feeding time for the animals as they are very aggressivetowards objects placed through the wire cage. The chewing process on thetube or string or other material is sufficient to deposit enough salivafor nucleic acid detection. Return visits may be required for animalsthat are not compliant.

One caution for this method is that the sampling tube or string or othermaterial may not touch the wire cage since environmental virus is likelyto be included in the sample. Care must be taken at this point to ensurethat no contamination results before the sample is safely placed in itslabeled sampling container. As taught before with respect to sampleacquisition for antibody testing (LFIA) and PCR testing, the samplinglid is opened and the portion of tube or string or other material is cutoff allowing it to fall into the container and then the lid is closed.Specific duties of each hand are practiced as described in Appendix “D”.

(3) Demonstration of Elimination of Pathogen From A Herd:

The success of the screening method taught in this patent document canclearly be seen by examination of FIG. 9 which extends the data of FIG.1 for the year 2009. It is immediately evident beginning in the latefall of 2008 that, after employing the testing and selection methodtaught in this patent document, the death rate had fallen at least tothe levels observed before the mADV outbreak, if not even lower. Thereason the death rate is never zero is due to the fact that some deathsnaturally occur due to environmental stress and other factors. However,the method taught herein has clearly been successful in eliminating themADV epidemic.

The method of the present invention has been exemplified by applicationto the elimination of mADV from a mink herd. The basic principles ofscreening using PCR detection of a pathogen's nucleic acid signature,with or without additional screening technologies such as antibodytesting (ELISA or LFIA) to identify and remove infected animals from aherd has general applicability to a wide range of animals. Thetechniques may even be extended to populations of wild animalsparticularly through the PCR testing of manure.

The discovery of primers that can identify the lethal mADV permits theassembly of testing kits that may be employed on mink farms. Simple kitsmay contain just the primers for mADV with the users supplying referenceprimers and laboratory facilities. More advanced kits may contain notonly the mADV primers but also the GAPDH or other internal referencemarker primers along with the remaining materials required to screen byPCR.

APPENDIX “A” DNA Extraction

Samples received for detection of mink Aleutian Disease Virus (mADV)were processed using RNase/DNase free microcentrifuge tubes and sterilepipette tips containing aerosol filters. Samples collected consisted of2 mL microcentrifuge tubes containing either:

1. Blood soaked cotton swab

2. Urine soaked cotton swab

3. Environmentally obtained sample on wetted cotton swab

4. Manure sample inside small diameter tube(s)

5. Placental manure sample inside small diameter tube(s)

6. Blood collected in heparinized glass or plastic capillary tube

7. Blood collected from pipette tip

8. Blood collected and dried onto Qiagen QIAcard

9. Saliva collected on applicator

Total DNA from cotton swab and small tube samples was extracted andpurified using Qiagen DNeasy Blood & Tissue Kit (Qiagen, Inc., Valencia,Calif.). The suggested manufacturer's protocol “Purification of TotalDNA from Animal Blood or Cells (Spin-Column)” was performed. Minorchanges were incorporated into the protocol for manure and placentalmanure samples. For samples containing more than one small tube, theMaster Lysis Buffer volume was increased two fold, samples were appliedto Spin Columns/Collection Tubes in 2 sequential loading applications(due to increased volume), 8000 rpm spins for 1 minute were increased to9000 rpm for 3 minutes, and 13600 rpm spin for 3 minutes increased to 6minutes. Total DNA from cotton swab, small diameter tube, capillarytube, pipette tip, QIAcard (excised 2.5 sq mm), and saliva applicatorsamples was extracted using Epicentre QuickExtract DNA ExtractionSolution (Epicentre Biotechnologies, Madison, Wis.). The suggestedmanufacturer's protocol was performed with the following changes: forcotton swab and small diameter tube the volume of QE used was 100 uL andfor capillary tube, pipette tip, QIAcard, and saliva applicator thevolume of QE used was 50 uL. The final solution was diluted 1:4 withDNase free water (Boston BioProducts Inc., Worcester, Mass.). PCRmethods including extraction methods and PCR techniques are undergoingrapid developments including advances in instrumentation. The processesdescribed above and below are currently practiced on the inventor'sfarm. However, these methods should not be considered limiting andadvanced PCR techniques can be employed in the overall method describedin this patent document.

APPENDIX “B” PCR Reaction Conditions

Extracted DNA, oligonucleotide primers, and GoTaq Green Master Mix(Promega Corporation, Madison, Wis.) were mixed together followingPromega's suggested protocol for PCR. Mineral oil was added to samplesbefore placing them in PerkinElmer 480 Thermocycler (PerkinElmer,Waltham, Mass.). Basically, the PCR steps included initial denaturation(95° C. for 2 minutes) followed by a 40 cycle loop of denaturation (95°C. for 30 seconds), annealing (see table below), and extension (72° C.for 1 minute), and then final extension (72° C. for 5 minutes) with ahold at 4° C. The following table summarizes primer and PCR conditions:

TABLE 4 Swab Multplex GAPDH ADV Anneal DNA Small Tube QE DNA:H2O GAPDH(uM) (uM) (° C.) (uL) DNA (uL) 1:4 (uL) V1a No — 0.1 57 3 — — V2 No —0.6 55 5 — — V3 Yes 0.2 0.4 57 10 — — V4 No — 0.1 55 5 — — V4b/V5b No —0.4 57 6 — — V5 Yes 0.2 0.4 57 10 5 5 V6a No — 0.1 57 10 — —

Completed PCR reactions were subjected to agarose electrophoresis. PCRproducts (amplicons) were visualized by UV fluorescence using GelRedNucleic Acid Stain (Phenix Research Products, Candler, N.C.)incorporated in the agarose. The presence of the GAPDH amplicon (250 bp)in the sample indicated that (cellular) DNA was extracted correctly andPCR performed properly. Appearance of the mADV amplicon (802 bp for V5)indicated the presence of viral DNA in sample.

APPENDIX “C” Cleaning/Sanitation

After removal of mink from the area, the first steps in cleaning aredescribed as “dry cleaning” whereupon any remaining feed, manure, andother debris is scrapped from the pens and used bedding materials areremoved from the boxes and allowed to fall to the ground. Next themanure, bedding and other materials are removed as much as possible andtaken to a compost pile outside and downwind from the ranch. Spreadingof this material is not recommended as virus may spread to feral animalsand perpetuate the infection outside of the farm. Layering of manure and“quick lime” (CaO) to this compost pile has been recommended to raisethe pH to unfavorable levels for the AD virus to survive.

If boxes are removable from their pens, they are immersed in a 3% NaOHsolution as well as any other wooden-ware associated. These are thencleaned typically with a cleaning machine delivering 4 GPM @ 3000 PSI @190 degrees F. The outside surfaces of the box are done first finishingwith the inside surfaces. Other parts are cleaned similarly whereuponthe box with its parts are removed from the shed and immersed in a 500ppm solution of Oxine (Bio-Cide International, Norman, Okla.) andpalletized in a way for air circulation for the natural drying of theOxine solution from the boxes. Afterward they are stretched wrapped forprotection and taken to clean storage until needed.

The next phase of cleaning addresses the wire pens and inside surfacesof the shed. In one method the pens are sprayed with a 3% NaOH solutionwith the optional addition of a foaming agent to enhance maximum contactto the extremely large surface area involved. While this is soaking, theinside roof and other areas are sprayed with a detergent [Complete Plus,(Camco Chemical, Madison, Wis.)], again with the optional use of afoaming agent. The 3% NaOH solution is not recommended on surfaces thatare aluminum such as shed roofs so the use of a detergent is usedinstead. Rinsing of the inner roof surface and other structural parts ofthe shed is preformed with the same machine initially before the wirepens are done working in a top to bottom fashion. The pens are carefullyrinsed in a manner that directs the spray to as many angles possible tominimize shadowed areas formed by the spraying action. The pens are thensprayed with a 500 ppm solution of Oxine and allowed to air dry. Againthe addition of a foaming agent enhances the contact time andcompleteness of the sanitizing solution. The final step of preparing theshed is to broadcast CaO inside and outside of the shed by use of agarden pulled lawn broadcaster. The CaO is applied at the rate ofapproximately six pounds per square yard. The shed remains in this stateuntil just prior to moving in PCR mADV negative animals. At that time,immediately before the shed is utilized, a second application of 500 ppmOxine is applied to the pens to ensure sanitation before use. Under allcircumstances, strict ranch hygiene is absolutely essential for thesuccessful implementation of eradication of the disease. Animal testingalone will not ensure elimination of a pathogen without adherence to thehighest levels of biosecurity.

APPENDIX “D” Blood Collection Process for Antibody (ELISA or LFIA) andPCR Testing

To minimize the transmission of the disease during this procedure, atechnique of using Oxine soaked handling gloves is employed as toprovide a sanitizing surface for any bodily fluids from the animals tobe neutralized upon contact. The gloves are soaked in a 500 ppm solutionof Oxine until saturated and the handler first dawns a pair of latexgloves before the soaked catching gloves to protect his/her hands fromthe long term exposure to the Oxine solution. The mink are carefullycaught as to avoid contact of the rear feet with the Oxine laden glovesas it was discovered that Oxine will produce a false positive reactionon LFIA test strips when incorporated with the blood sample (personalcommunication).

The handler holds the animal horizontal with the rear feet to him/herand extended beyond the pen with the fore feet placed firmly on the toppart of the pen while gently rolling the animal to the left side toraise the right rear foot upward. The sampling person prepares toacquire the blood sample. Since a third hand is required, the mouth ofthe sampler may be used to hold the stem ends of the sterile cottonswabs while the right hand holds the clippers and is the only hand usedto open and close sample containers. Reproducible non-crosscontaminating sample acquisition is crucial at this stage. It isimperative that the sampler maintains a clean hand, usually the mostdexterous one, and a sampling hand, one that is in repetitive physicalcontact with the animals. The two hands never exchange duties andmaintain their respective operations.

The technique of blood collection is best preformed as follows. With theleft hand, the sampler firmly grabs the elevated right rear foot of themink such that the foot pad rests completely on the left thumb of thesampler. With the right hand, the sampler skillfully clips a toenail,preferably from the smallest, last digit, just above the quick line witha small pair of toenail clippers maintaining the grip with his leftthumb and left fore finger of the left hand. Blood will flow momentarilyor, if not, a second clipping may be required or a slight relaxation ofthe grip may allow the flow of blood to proceed. The sampler removesfrom his mouth a sterile cotton swab with his clean right hand andacquires first the sample for blood LFIA. The stem of the swab istransferred to the released left hand, the pre-labeled sample containerlid is opened with the thumb and fore finger of the right hand and thecotton head of the blood soaked swab is cut with the clippers still heldin the right hand just above the cotton head. The lid is closed with theright thumb and fore finger. The stem of the swab is discarded with theleft hand and is then used to re-grip the animal's right rear foot asbefore. Secondly, the sample for blood PCR is acquired in the samefashion excluding any contact with anything other than free flowingblood from the toenail to avoid environmental virus contamination. Thisprocess is repeated using the same hands in the same fashion aspreviously described. Upon completion of acquiring samples from theanimal, the clippers are wiped free of any blood with a paper towelusing the left hand and exchanged with a second pair of clippers soakingin 500 ppm of Oxine. This second pair is carefully dried with a cleanportion of paper towel using the left hand but not allowing the samplingfingers to touch any part of the clipper's cutting surface. Layers ofclean towel are maintained between the left fingers and cutting surfaceand the handles are held by the right hand. The purpose of this dryingaction is to eliminate false positive LFIA that may arise with Oxinepresent in the blood sample. In practice, the used towel is notdiscarded until used to remove blood from the next clipping action priorto immersion in Oxine. A fresh towel is only used for the pair ofclippers immediately removed from the Oxine.

Blood collection can also be taken using 1.0 to 1.1 mm ID Na heparinizedplastic capillary tubes commonly used in CIEP(counterimmunoelectrophoresis) testing, (Globe Scientific, Paramus,N.J.). The mink is similarly handled and hygiene observed as above onlythe use of a capillary tube instead of cotton swab acquires the sample.By this method, volumes of samples can be accurately established due tothe constant capillary diameter and length of tube filled. For instancea half-filled capillary tube is approximately 35 uL in volume. In somesampling procedures, the contents of the capillary tube are expelled bythe use of a capillary bulb into a pre-labeled/bar coded sampling vialwith a snap top or into a pre-labeled/bar coded 48 or 96 well platesuitable for extraction and/or PCR.

Yet another collection process that has been successfully used is thespotting, spreading, and drying of a drop of blood onto a QIAcard(QIAGEN, Valencia, Calif.) and is useful for sample archiving. Punchedout portion of the dried, spotted area yields sufficient sample foranalysis and it has been found that cross-contamination is not a factorto be considered by the protocol outlined by the manufacturer. Samplesare stored at −20° C. until ready for testing for PCR and LFIA samplesare stored at 4° C. until testing.

APPENDIX “E” Environmental Collection Process for PCR

Unlike other testing methods currently available, the use of PCRtechnology allows testing of the environment for mADV presence. This isparticularly important to eliminate the possibility of recontaminationof the animals that are returned to the pens. Thus, environmentalsampling is most useful after a cleaning procedure to determine theefficacy of the cleaning and sanitizing processes. Typically the methodused is as follows. An area to be investigated is aggressively rubbedwith a cotton swab that has been soaked in a Phosphate Buffered Saline(PBS, Boston BioProducts, Inc., Worcester, Mass.). The presoaking of thecotton swab aids in the acquisition and preservation of the sample. Thesampling area can include, but is not limited to, the wire cages, woodenboxes and their parts, inside of the housing roof surfaces, and theground to name a few of the more obvious and worthwhile sites. Aspreviously stated, the use of proper hygiene while manipulating thesample is always important. The sample may be stored at 4° C. until thePCR process.

We claim:
 1. A method to eliminate Aleutian Disease Virus (mADV)infection in farmed mink populations comprising the following steps: a)visually inspecting the minks for observable signs of infection; b)removing from the population those minks showing observable signs ofinfection; c) obtaining and testing the urine of the remaining minks forantibodies specific for mADV proteins indicating infection with thevirus; d) removing from the population those minks having antibodies intheir urine indicating infection with the virus; e) obtaining blood orsaliva samples of the remaining minks f) extracting DNA from the bloodor saliva samples; g) assaying the DNA from the blood or saliva samplesfor mADV using PCR amplification performed with one or more of thefollowing primer sets, or their complementary sets, or sets having atleast 85% homology thereto: SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9and SEQ ID NO: 10; and SEQ ID NO: 11 and SEQ ID NO: 12 to determinewhether the animals are infected with mADV; and h) removing from thepopulation those minks in which the PCR assay of the blood or salivaindicates infection with the virus.
 2. The method of claim 1 in whichantibody testing is performed either by (ELISA) enzyme-linkedimmunosorbent assay or with a (LFIA) lateral flow immuno assay strip. 3.The method of claim 1 further comprising the removal of minks in whichan initial PCR assay for the pathogen in the mink's blood or saliva didnot detect the presence of the pathogen but in which a PCR assay retestat a later time detects the presence of the virus in the mink's blood orsaliva.
 4. The method of claim 1 further comprising the removal of minksin which a PCR assay for mADV in the mink's birthing manure detects thepresence of the mADV virus.
 5. The method of claim 1 further comprisingusing one or more primer pairs that serve as either negative or positivecontrols to verify the functioning of the (PCR) polymerase chainreaction.
 6. The method of claim 5 in which the primer pairs that serveas either negative or positive controls are the primer pairs formammalian (GAPDH) glyceraldehyde 3-phosphate dehydrogenase.
 7. A methodto eliminate Aleutian Disease Virus (mADV) infection in farmed minkpopulations comprising the following steps: a) visually inspecting theminks for observable signs of infection; b) removing from the populationthose minks showing observable signs of infection; c) obtaining andtesting the urine of the remaining minks for antibodies specific formADV proteins indicating infection with the virus; d) removing from thepopulation those minks having antibodies in their urine indicatinginfection with the virus; e) obtaining blood or saliva samples of theremaining minks; f) extracting DNA from the blood or saliva samples; g)assaying the DNA from the blood or saliva samples for mADV using PCRamplification performed with one or more of the following primer sets,or their complementary sets, or sets having at least 85% homologythereto: SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10;and SEQ ID NO: 11 and SEQ ID NO: 12 to determine whether the animals areinfected with mADV; h) removing from the population those minks in whichthe PCR assay of the blood or saliva indicates the presence of thepathogen; i) obtaining a birthing manure sample from each subsequentlitter of the remaining animals after the females have consumed theplacentas of the offspring; j) extracting DNA from the manure sample;and k) assaying the DNA from the birthing manure sample for mADV usingPCR amplification performed with one or more of the following primersets, or their complementary sets, or sets having at least 85% homologythereto: SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO: 10;SEQ ID NO: 11 and SEQ ID NO: 12 and l) removing from the populationthose minks in which the PCR assay for the virus in the animal'sbirthing manure detects the presence of the virus.
 8. A method toeliminate mADV infection in farmed mink populations comprising thefollowing steps: a) obtaining a birthing manure sample from each litterafter consumption by the female of the placentas of her offspring; b)extracting DNA from the manure sample; c) assaying the DNA from thebirthing manure sample for mADV using PCR amplification performed withone or more of the following primer sets, or their complementary sets,or sets having at least 85% homology thereto: SEQ ID NO: 7 and SEQ IDNO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12;d) identifying those minks from litters where the manure assayed for thepresence of mADV; e) determining the infected or non-infected status ofeach mink further comprising the following steps: 1) obtaining blood orsaliva samples of each offspring and the female parent; 2) extractingDNA from the blood or saliva samples; and 3) assaying the DNA from theblood or saliva samples for mADV using PCR amplification performed withone or more of the following primer sets, or their complementary sets,or sets having at least 85% homology thereto: SEQ ID NO: 7 and SEQ IDNO: 8; SEQ ID NO: 9 and SEQ ID NO: 10; SEQ ID NO: 11 and SEQ ID NO: 12f) removing from the population those minks in which a PCR assay formADV in the mink's blood or saliva samples detects the presence of thevirus.
 9. A method to eliminate Aleutian Disease Virus (mADV) infectionin farmed mink populations comprising the following steps: a) visuallyinspecting the minks for observable signs of infection; b) removing fromthe population those minks showing observable signs of infection; c)obtaining and testing the urine of the remaining minks for antibodiesspecific for mADV proteins indicating infection with the virus; d)removing from the population those minks having antibodies in theirurine indicating infection with the virus; e) obtaining blood or salivasamples of the remaining minks f) extracting DNA from the blood orsaliva samples; g) assaying the DNA from the blood or saliva samples formADV using PCR amplification performed with one or more of the followingprimer sets, or their complementary sets, or sets having at least 85%homology thereto: SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ IDNO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 13 and SEQ ID NO: 14 todetermine whether the animals are infected with mADV; and h) removingfrom the population those minks in which the PCR assay of the blood orsaliva indicates infection with the virus.
 10. A method to eliminateAleutian Disease Virus (mADV) infection in farmed mink populationscomprising the following steps: a) visually inspecting the minks forobservable signs of infection; b) removing from the population thoseminks showing observable signs of infection; c) obtaining and testingthe urine of the remaining minks for antibodies specific for mADVproteins indicating infection with the virus; d) removing from thepopulation those minks having antibodies in their urine indicatinginfection with the virus; e) obtaining blood or saliva samples of theremaining minks; f) extracting DNA from the blood or saliva samples; g)assaying the DNA from the blood or saliva samples for mADV using PCRamplification performed with one or more of the following primer sets,or their complementary sets, or sets having at least 85% homologythereto: SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4;SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 13 and SEQ ID NO: 14 todetermine whether the animals are infected with mADV; h) removing fromthe population those minks in which the PCR assay of the blood or salivaindicates the presence of the pathogen; i) obtaining a birthing manuresample from each subsequent litter of the remaining animals after thefemales have consumed the placentas of the offspring; j) extracting DNAfrom the manure sample; and k) assaying the DNA from the birthing manuresample for mADV using PCR amplification performed with one or more ofthe following primer sets, or their complementary sets, or sets havingat least 85% homology thereto: SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO:3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 13 and SEQID NO: 14 and l) removing from the population those minks in which thePCR assay for the virus in the animal's birthing manure detects thepresence of the virus.
 11. A method to eliminate mADV infection infarmed mink populations comprising the following steps: a) obtaining abirthing manure sample from each litter after consumption by the femaleof the placentas of her offspring; b) extracting DNA from the manuresample; c) assaying the DNA from the birthing manure sample for mADVusing PCR amplification performed with one or more of the followingprimer sets, or their complementary sets, or sets having at least 85%homology thereto: SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ IDNO: 10; SEQ ID NO: 11 and SEQ ID NO: 12; d) identifying those minks fromlitters where the manure assayed for the presence of mADV; e)determining the infected or non-infected status of each mink furthercomprising the following steps: 1) obtaining blood or saliva samples ofeach offspring and the female parent; 2) extracting DNA from the bloodor saliva samples; and 3) assaying the DNA from the blood or salivasamples for mADV using PCR amplification performed with one or more ofthe following primer sets, or their complementary sets, or sets havingat least 85% homology thereto: SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO:3 and SEQ ID NO: 4; SEQ ID NO: 5 and SEQ ID NO: 6; SEQ ID NO: 13 and SEQID NO: 14 f) removing from the population those minks in which a PCRassay for mADV in the mink's blood or saliva samples detects thepresence of the virus.